Seminal Research​In Vitro Analysis of The Interaction Between Yeast Fimbrin And Actin:A Model for A Mechanism of Allele-Specific Suppression​Dr. Tanya Sandrock​

​Dr. Tanya SandrockTitle: President, Integratech Proteomics, LLCResearch Topic: In Vitro Analysis of The Interaction Between Yeast Fimbrin And Actin: A Model for A Mechanism of Allele-Specific SuppressionInstitution: The University of ArizonaCommittee Chair: Dr. Carol -DireckmatinAbstract: The actin cytoskeleton in yeast plays a role in many morphological events such as cell growth, secretion, polarity, bud emergence, and endocytosis (125). Some proteins have been identified that regulate the elongation, nucleation, and stability of the actin cytoskeleton (101). One of the many proteins controlling the state of the actin cytoskeleton is an actin bundling protein encoded by the gene SAC6 (suppressor of actin). In this study, I have examined some of the phenotypic consequences of overexpression of Sac6p and analyzed interactions between Sac6p and actin in vitro. In Chapter 3,1 describe the purification of Sac6p and some initial characterization of the protein. The purification scheme described in Chapter 3 was used to analyze the potential biochemical mechanism of suppression seen between mutant sac6 and act1 alleles. This portion of the study is described in Chapter 4. I have also investigated the molecular basis of allelc-specific suppression by analyzing the interaction between wildtype and mutant actin and wild-type and mutant Sac6p. The biochemical assays show in vitro suppression of binding and crosslinking activity between Sac6 suppressor protein and mutant actin protein. In addition, the Sac6 suppressor proteins tested have an increased affinity to wild-type actin. This analysis, as well as previous genetic data, is consistent with the idea that suppression results from an overall increase in affinity to actin rather than a strict "lock and key" mechanism previously hypothesized.

Overexpression of Sac6p under the conurol of a galactose inducible promoter results in growth inhibition, accumulation of multinucleated cells, and altered actin 12 cytoskeletal organization. To better understand why overexpression of Sac6p has detrimental effects, I devised a screen to isolate genomic mutations that can suppress the growth defects resulting from elevated levels of Sac6p. I have found that an actl-3 su-ain is able to suppress Sac6p overexpression and one of the mutants isolated in the screen is also an acti mutant allele. In addition, the temperature sensitive and osmotic sensitive phenotypes are not complemented by an actl-3 strain. These results suggest that the lethality is mediated through the interaction of Sac6p with actin. This study will be outlined in Chapter 5.

In the first chapter, I will first describe the yeast actin cytoskeleton, including some of the players identified that are needed to maintain the integrity of the cytoskeleton. In the second part of the introduction I will summarize a number of studies that employ allele specific suppression and the models of suppression that have been hypothesized from these studies.